Stem cells are cells found in all multicellular organisms. They retain the ability to renew themselves through mitotic cell division and can differentiate into a diverse range of specialized cell types. The classical definition of a stem cell requires that it possess two properties, self-renewal and potency. Self-renewal is defined as the ability to go through numerous cycles of cell division while maintaining an undifferentiated state, while potency is the capacity to differentiate into specialized cell types. In the strictest sense, this requires stem cells to be either totipotent or pluripotent, i.e., to be able to give rise to any mature cell type, although multipotent or unipotent progenitor cells are sometimes referred to as stem cells
The two broad types of mammalian stem cells are embryonic stem cells, which are found in blastocysts, and adult stem cells, which are found in adult tissues.
Embryonic stem (ES) cells are cells derived from the epiblast tissue of the inner cell mass (ICM) of a blastocyst or earlier morula stage embryos. ES cells are pluripotent and give rise during development to all derivatives of the three primary germ layers, i.e., ectoderm, endoderm, and mesoderm. Thus, ES cells can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type.
A human embryonic stem cell is defined by the presence of several transcription factors and cell surface proteins. The transcription factors, Oct4, Nanog, and SOX2, form the core regulatory network that ensures the suppression of genes that lead to the differentiation and maintenance of pluripotency.
An adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ. An adult stem cell can renew itself and can differentiate to yield the major specialized cell types of the tissue or organ. The primary role of adult stem cells in a living organism is to maintain and repair the tissue in which they are found. A great deal of adult stem cell research has focused on studying their capacity to divide or self-renew indefinitely and their differentiation potential. In mice, pluripotent stem cells are directly generated from adult fibroblast cultures.
While embryonic stem cell potential remains untested, adult stem cell treatments have been used for many years to successfully treat leukemia and related bone/blood cancers through bone marrow transplants. The use of adult stem cells in research and therapy is not as controversial as embryonic stem cells, because the production of adult stem cells does not require the destruction of an embryo.
Meanwhile, during mammalian embryo development, initial cellular differentiation becomes readily observable during compaction and blastocyst formation. At that time, the ES cells become committed to two distinct developmental pathways, i.e., the trophectoderm (TE), giving rise to extraembryonic tissues, and the inner cell mass (ICM), giving rise to the definitive germ layers of the embryo. This process of cellular differentiation is characterized by distinct alterations in gene and protein expression, including transcription factors involved in the determination of cell fate, cytokines involved in autocrine and paracrine signaling, and other structural and functional proteins involved in cell morphology and physiology.
An increasing number of transcription factors that are involved in the determination of cell fate at this key point in early embryonic development have been identified. Two of these transcription factors, Oct4 and Nanog, are thought to work in concert to maintain pluripotency and self-renewal in ICM and ES cells.
Oct4, a POU octamer-binding domain transcription factor, is known to be critical in mammalian embryonic development. Oct4 protein is expressed at the early blastocyst stage in both ICM and TE. However, expression is rapidly down-regulated in the TE and is generally limited to the ICM cells by the expanded blastocyst stage.
It has been demonstrated that Oct4 plays a pivotal role in establishing and maintaining cell lineage pluripotency, both in vivo and in vitro. The deletion of Oct4 causes early lethality in mouse at 3.5 days of gestation. A pluripotent ICM is not formed and the cells differentiate into a TE lineage. Conditional repression of Oct4 in mouse ES cells also resulted in differentiation into trophoblast lineage, while overexpression resulted in differentiation into primitive endoderm. These studies suggested that the level of Oct4 expression was a critical factor in the determination of cell lineage. It has also been proposed that Oct4 is necessary for the maintenance of ICM pluripotency and acts, in part, by repressing trophoblast lineages in the mouse.
The expression of a novel homeobox gene, Nanog, during early embryogenesis in the mouse has been reported. Nanog also plays a key role in self-renewal and the maintenance of pluripotency in mouse ICM and ES cells. Deletion of the gene for Nanog is an embryonic lethality and results in the loss of pluripotency in both ICM and ES cells. Nanog deficient ICM (Nanog -/-) and ES cells differentiate into extraembryonic endoderm. Nanog protein was detected as early as the morula stage. Strikingly, Nanog was strongly expressed in the inner apolar cells, but weakly or not expressed in the outer polar cells of the late morula. At the blastocyst stage, Nanog was only expressed in the ICM and was not expressed in the TE.
In sum, Oct4 and Nanog are essential factors for self-renewal and pluripotency of ICM and ES cells. Based on these facts, the present inventors have aimed to establish stem cells maintaining self-renewal potential and pluripotency in the undifferentiated state. As a result, the present inventors have developed a method of increasing self-renewal and suppressing differentiation of stem cells by treating them in combination with Nanog and Oct4, which are genetically engineered to have cell permeability.